There are currently seven P2X receptor subunits (P2X_1–7) defined by molecular cloning. The functional identification of these receptors has relied primarily on the potency of α,β-methylene-ATP relative to that of ATP and on the kinetics of receptor desensitization. In the present experiments we found that the 2′,3′-O-(2,4,6-trinitrophenyl)-substituted analogs of ATP are selective and potent antagonists at some but not all P2X receptors. The trinitrophenyl analogs of ATP, ADP, AMP, and GTP produced a reversible inhibition of ATP-evoked currents in human embryonic kidney 293 cells expressing P2X₁ receptors, P2X₃ receptors, or both P2X₂ and P2X₃ (heteromeric) receptors; IC₅₀ values were close to 1 nm. These compounds were at least 1000-fold less effective in blocking currents in cells expressing P2X₂, P2X₄, or P2X₇receptors (P2X₅ and P2X₆ not tested). GTP, 2,4,6-trinitrophenol, and the 2′,3′-trinitrophenyl analog of adenosine (0.1–10 μm) had no effect. Thus, we have identified a structural motif that confers antagonist action at P2X receptors that contain P2X₁ or P2X₃ subunits (the α,β-methylene-ATP-sensitive subclass).